KR101249801B1 - Compositions for stem cell culture media and method for culturing the stem cell using the compositions - Google Patents

Abstract

The present invention relates to a stem cell maintenance composition comprising a melatonin receptor agonist in a basic medium. The present invention also relates to a medium composition for inducing stem cell differentiation comprising a melatonin receptor antagonist in a basic medium. By using the medium composition, stem cells may be cultured or stem cells may be differentiated while maintaining the characteristics of stem cells.

Stem Cells, Stem Cellity, Differentiation, Stem Cell Culture Media

Description

Stem cell culture composition and stem cell culture method {COMPOSITIONS FOR STEM CELL CULTURE MEDIA AND METHOD FOR CULTURING THE STEM CELL USING THE COMPOSITIONS}

The present invention relates to a medium composition and stem cell culture method used for stem cell culture.

Stem cells are cells that remain undifferentiated into specific cells and have the ability to differentiate into all kinds of cells constituting the organism, such as nerves, blood, and cartilage, if necessary.

There are two ways to obtain these stem cells. The first is from embryos derived from fertilized eggs (embryonic stem cells), and the second is to recover stem cells (adult stem cells) stored in each part of the adult body. Although functionally different, embryonic stem cells and adult stem cells are characterized by differentiation into various cell types.

Human embryonic stem cells have a pluripotency that is capable of producing all the cells of our body, and have the self-renewal ability to produce cells that resemble themselves indefinitely. These two properties together are called "stemness".

Several signaling systems involved in the stem cell maintenance of human embryonic stem cells are currently identified. The representative ones are Fibroblast Growth Factor (FGF) signaling pathway and TGF-Beta (Transforming Growth Factor-Beta) / Activin signaling system. In addition, transcription factors in the cytoplasm, such as Octamer Binding Transcription Factor-4 (OCT4), Nanog, and Sox2 (SRY Determining Region-Y) Box-2), are known to play an important role (Biswas A, Hutchins). R., Stem Cells Dev . 2007 (2), 213-22; Darr H, Benvenisty N., Regen Med . 2006 (3), 317-25).

In this regard, the components of these signaling systems, SMAD 2/3 (Sma and MAD (Mothers Against Decapentaplegic) Related Protein-2 / 3), ALK4 (Activin Receptor-Like Kinase-4), and ALK5 (Activin Receptor-Like Kinase) -5), ALK7 (Activin Receptor-Like Kinase-7), Nodal, Cripto, LEFTY1 (Left-right determination factor-1) and LEFTY2 (Left-right determination factor-2) also maintain stem cells of human embryonic stem cells (Xiao L, Yuan X, Sharkis SJ., Stem Cells . 2006 (6), 1476-1486; Besser D., J Biol Chem . 2004 (43), 45076-45084; and James D, Levine AJ, Besser D, Hemmati-Brivanlou A., Development . 2005, 132 (6), 1273-1282).

In addition, WNTs (Wingless-Type MMTV Integration Site Family Members) proteins and WNT signaling systems have also been shown to play an important role in maintaining the stem cell function of human embryonic stem cells. Inhibition of GSK-3b (Glycogen Synthase Kinase-3b), a component of this signaling system, and subsequent increase in beta-catenin have been reported to play an important role (Miyabayashi T, et . al , Proc Natl Acad Sci USA . 2007, 104 (13), 5668-5773).

In addition, S1P (Sphingosine-1-Phosphate) is also involved in stem cell maintenance through EDG (Endothelial Differentiation Gene) receptors and its associated G-proteins, or by signaling systems through the Platelet Derived-Growth Factor (PDGF). (Chase LG, Firpo MT., Curr Opin Chem Biol . 2007 (4), 367-372; and Inniss K, Moore H., Stem Cells Dev . 2006 (6), 789-796.).

A method currently commonly used for culturing stem cells, particularly human embryonic stem cells, is co-culture using mouse embryonic fibroblasts as feeder cells. This is a method in which feeder cells supply the factors necessary for culturing while maintaining the stem cell properties of human embryonic stem cells. To date, it is not known what factors are provided in the feeder cells are important for the stem cell maintenance of human embryonic stem cells.

Human embryonic stem cells cultured through coculture with mouse embryonic fibroblasts have many difficulties in clinical use. This is because there is a possibility of transmission by xenogen between different species. Therefore, attempts have been made to culture human embryonic stem cells in the absence of feeder cells. However, until now, a method of culturing human embryonic stem cells stably using a feeder-free culture medium in which animal-derived substances have been completely removed and an extracellular matrix of a culture vessel has not been established. have.

Therefore, the most important issue to be solved at present is to make a medium containing no animal-derived material that can culture human embryonic stem cells for a long time without using feeder cells. To this end, it is important to identify signaling systems and ligand substances involved in stem cell growth of human embryonic stem cells and to understand the mechanism of stem cell maintenance.

However, it is thought that there are signaling systems and ligands that are important for maintaining stem cell traits that have not yet been identified. Therefore, the inventors of the present invention continued to find a substance involved in the signaling system involved in stem cell maintenance. On the contrary, the study was also conducted on substances that inhibit these signaling systems and induce differentiation of stem cells. As a result, the present inventors have found a substance that maintains stem cellity and a substance that induces stem cell differentiation and have reached the present invention.

An object of the present invention is to provide a stem cell maintenance medium composition and a stem cell differentiation induction medium composition.

Another object of the present invention is a method for culturing stem cells while maintaining the characteristics of stem cells using the stem cell maintenance medium composition; It is to provide a method for inducing differentiation of stem cells using the stem cell differentiation induction medium composition.

Still another object of the present invention is to provide a culture medium for infection-free and feeder-free culture comprising the medium composition.

According to an aspect of the present invention, the present invention provides a stem cell maintenance medium composition comprising a melatonin receptor agonist in the basal medium.

The basal medium may be used as a medium for stem cell culture, without limitation as long as the medium is well known to those skilled in the art. The base medium may be prepared by artificially synthesizing, and a commercially prepared medium may be used. Examples of commercially prepared media include Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, α-MEM (α-Minimal). essential medium (G-MEM), Glasgow's Minimal Essential Medium (G-MEM), Isocove's Modified Dulbecco's Medium (IMDM), MEF and mTeSR-1, but are not limited thereto.

In the present invention, the following two small molecule compounds search methods were performed to find a compound that maintains stem cell activity and a compound that inhibits stem cell maintenance and induces differentiation of stem cells.

First, it is a method to search for small molecule compounds involved in stem cell maintenance. Human embryonic stem cells are applied to a culture vessel coated with an extracellular matrix called “Matrigel (BD Biosciences, USA), and then put into differentiation induction medium to induce spontaneous differentiation. The mixture was treated. On the last day of culture, human embryonic stem cell colonies were formed, colony-shaped observation, alkaline phosphatase staining, and various kinds of undifferentiated markers (Oct4, SSEA4, Tra1-60, Tra1-81, etc.) Immunostaining of these plants is used to identify small molecule compounds that continue to maintain stem cellity (ie, inhibit differentiation).

The second method is to find small molecule compounds that inhibit stem cells, that is, induce differentiation of stem cells. Human embryonic stem cells were applied to a culture vessel coated with Matrigel, and then treated with small molecule compounds while being cultured using an undifferentiated maintenance culture medium (eg, mTeSR-1 (Stemcell Technologies, USA)). On the last day of culture, human embryonic stem cells were observed for colony formation and colony formation, alkaline phosphatase staining, and various types of undifferentiated markers (Oct4, SSEA4, Tra1-60, Tra1-81). Immunostaining finds small molecule compounds that inhibit stem cell proliferation of human embryonic stem cells.

As a result, it was found that the melatonin receptor agonist is involved in maintaining the stem cellularity of human embryonic stem cells, while the melatonin receptor antagonist inhibits the stem cell maintenance and induces differentiation.

The melatonin receptor agonists include all melatonin receptor antagonists already known in the art. Specific examples include N- [2- (5-methoxy-1 H-indol-3-yl) ethyl] acetamide (N- [2- (5-methoxy-1 H-indol-3-yl) ethyl] acetamide); N- [2- (2-iodo-5-methoxy-1H-indol-3-yl) ethyl] acetamide (N- [2- (2-iodo-5-methoxy-1H-indol-3-yl ) ethyl] acetamide); N-[(2R) -2- (6-chloro-5-methoxy-1H-indol-3-yl) propyl] acetamide (N-[(2R) -2- (6-chloro-5-methoxy- 1H-indol-3-yl) propyl] acetamide); N-butanoyl-2- (2-methoxy-6H-isoindolo [2,1-a] indol-11-yl) ethanamine (N-butanoyl-2- (2-methoxy-6H-isoindolo [2 , 1-a] indol-11-yl) ethanamine); 3- (1-acetyl-3-methyl-piperidine) -5-methoxyindole (3- (1-acetyl-3-methyl-piperidine) -5-methoxyindole) and N- {2- [3- ( 3-aminophenyl) -7-methoxy-1-naphthyl] ethyl} acetamide (N- {2- [3- (3-aminophenyl) -7-methoxy-1-naphthyl] ethyl} acetamide) It may be selected from the group, but is not necessarily limited thereto.

In the present invention, the melatonin receptor agonist may be used by including 0.001 to 1000 uM in the medium composition. If the content of the melatonin receptor agonist is less than 0.001 uM may cause a problem that the stem cell maintenance effect does not appear. On the other hand, when the content of the melatonin receptor agonist exceeds 1000 uM, there may occur a problem that the cytotoxicity appears.

Stem cells cultured through the stem cell maintenance culture composition may be embryonic stem cells or adult stem cells.

In addition, the adult stem cells may be derived from adult stem cells of all tissues. For example, adult stem cells are composed of neural stem cells, liver stem cells, hematopoietic stem cells, cord blood stem cells, epidermal stem cells, gastrointestinal stem cells, endothelial stem cells, muscle stem cells, mesenchymal stem cells, and pancreatic stem cells. It may be selected from the group, but is not necessarily limited thereto.

Currently used feeder-free culture methods include animal derived materials. Obtaining clinical human embryonic stem cells requires a new culture medium that is completely free of these animal-derived materials.

The stem cell maintenance medium composition according to the present invention is for infectious agents-free (xenogen-free) and feeder-free culture that can culture embryonic stem cells without using feeder cells It can be used to make culture medium.

Specifically, by using materials involved in stem cell maintenance of human embryonic stem cells, such as the aforementioned melatonin receptor agonist, it is possible to prepare a feeder-free and infection-free medium composition, and through this medium composition Embryonic stem cells can be cultured.

In addition, when human-derived cells are used as feeder cells instead of mouse embryonic fibroblasts, clinical human embryonic stem cells may be cultured only by using a new infection-free medium instead of a medium containing an existing animal-derived material. The above-mentioned melatonin receptor agonists can also be used to develop such media.

In another aspect of the invention, the present invention provides a stem cell differentiation-inducing medium composition comprising a melatonin receptor antagonist in the basal medium.

In the present invention, the melatonin receptor antagonist may be used by including 0.001 to 1.000 uM in the medium composition. If the content of the melatonin receptor antagonist is less than 0.001 uM, there may be a problem that the stem cell differentiation induction effect does not appear. On the other hand, if the content of the melatonin receptor antagonist exceeds 1000 uM, there may occur a problem appearing cytotoxicity.

The melatonin receptor antagonist includes all melatonin receptor antagonists known in the art. In the present invention, specific examples include N-butanoyl 2- (5,6,7-trihydro-11-methoxybenzo (c) cyclohept [2,1-a] indol-13-yl) ethanamine (N- butanoyl 2- (5,6,7-trihydro-11-methoxybenzo (c) cyclohept [2,1-a] indol-13-yl) ethanamine); N- (4-phenyltetralin-2-yl) propanamide (N- (4-phenyltetralin-2-yl) propanamide); 5-hydroxyethoxy-N-acetyltryptamine; N- [2- (2-benzyl-1H-indol-3-yl) ethyl] acetamide (N- [2- (2-benzyl-1H-indol-3-yl) ethyl] acetamide); N- (2-naphthalen-1-ylethyl) cyclobutanecarboxamide (N- (2-naphthalen-1-ylethyl) cyclobutanecarboxamide) and N- (2- {7- [3-({8- [2- Acetylamino) ethyl] -2-naphthyl} oxy) propoxy] -1-naphthyl} ethyl) acetamide (N- (2- {7- [3-({8- [2-acetylamino) ethyl]- 2-naphtyl} oxy) propoxy] -1-naphthyl} ethyl) acetamide), but is not necessarily limited thereto.

The type of basic medium and stem cells used in the stem cell differentiation-inducing medium composition is the same as mentioned above.

Differentiation into specific cells is essential for the development of cell therapies using stem cells. If a certain number of embryonic stem cells remain undifferentiated and are present, they are more likely to form "teratomas" after transplantation in vivo. If melatonin receptor antagonists are used to further promote differentiation, more differentiated cells will be obtained and the incidence of cancer will be much reduced.

In addition, these melatonin receptor antagonists are characterized by inducing differentiation of stem cells. Therefore, the use of melatonin receptor antagonists can induce differentiation of stem cells into cells of a specific lineage or a specific type, and as a result, there is an advantage of efficiently obtaining these specific cells.

As mentioned above, melatonin receptor antagonists can be used to prepare media compositions in feeder-free and infection-free states, which can induce differentiation of embryonic stem cells.

In another aspect of the present invention, the present invention provides a method for culturing stem cells while maintaining the characteristics of stem cells using a stem cell maintenance medium composition comprising a melatonin receptor anti-inflammatory agent.

In another aspect of the invention, the invention provides a method of inducing differentiation of stem cells using a medium composition comprising a melatonin receptor antagonist.

The culture composition for infection-free and / or feeder-free may be made using a medium composition comprising a melatonin receptor agonist or a medium composition comprising a melatonin receptor antagonist according to the present invention. In addition, by using the culture medium comprising the medium composition according to the present invention can maintain the stem cell nature or promote the differentiation of stem cells can be obtained at the desired time the desired specific cells.

Hereinafter, the present invention will be described in detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Preparation of stem cell maintenance medium composition and stem cell undifferentiation confirmation

Each well of a 96 well plate coated with Matrigel (BD Biosciences, USA) was coated with pieces of colonies of human embryonic stem cells (CHA6 cell line, CHA University, Korea). Then, MEF medium (DMEM high-glucose (Welgene) + 10% FBS (Welgene) + 1% pennicillin / streptomycin (Caisson) + 1% NEAA (GIBCO) + 0.1% b-mercaptoethanol (GIBCO)) Human embryonic stem cell culture was started by putting it in (37.0 ° C., 5.0% CO ₂ ). At this time, embryonic stem cells were put in 5 pieces of chunks per well of 96 well plate. Then, 1 um of melatonin (N- [2- (5-methoxy-1H-indol-3-yl) ethyl] acetamide) (Sigma), a previously mentioned melatonin receptor agonist, was added daily for about 5-7 days The experiment was carried out to culture embryonic stem cells by adding to a new medium.

Human stem cells as a control medium for differentiation inhibition (FIG. 1 (a)) MEF medium without melatonin (FIG. 1 (b)), and MEF medium containing the differentiation promoter retinoic acid (Fig. Culture in 1 (c)).

Alkaline phosphatase staining was performed as follows. First, the cells were fixed with 4% paraformaldehyde (para-formaldehyde) for 1-2 minutes and washed once with 1x TBST (20 mM Tris-HCl, pH 7.4, 0.15M NaCl, 0.05%, Tween-20). Fast red violet, naphtol and H ₂ O were mixed 2: 1: 1 and treated for 15 minutes, washed once with 1x TBST and ground with 1x PBS.

As a result, as shown in Figure 1 (b), when using the MEF medium, the cells are differentiated and spread. On the other hand, when treated with 1 uM of melatonin, cells were less spread, maintaining some form of colony, and the expression of alkaline phosphatase, an undifferentiated label, was significantly increased (FIG. d)). In other words, it can be seen that melatonin plays a role in inhibiting the differentiation of stem cells and maintaining an undifferentiated state. Human embryonic stem cells are almost differentiated in the case of medium (MEF medium) which is not added melatonin or medium (MEF + RA) to which differentiation-inducing substance is added (FIG. 1 (b) and (c)). ).

Example 2 Preparation of Stem Cell Differentiation Media Composition and Confirmation of Stem Cell Differentiation

Each well of a 96 well plate coated with Matrigel (BD Biosciences, USA) was coated with pieces of colonies of human embryonic stem cells (CHA6 cell line, CHA University, Korea). After that, mTeSR-1 (Stemcell Technologies, USA: medium containing bFGF, TGFb, LiCl, GABA, pipecolic acid), which is an undifferentiated maintenance culture medium, was added to start culturing human embryonic stem cells (37.0 ° C, 5.0%). CO ₂ ). Embryonic stem cells were then put in 5 pieces of chunks in each well of a 96 well plate.) N-butanoyl 2- (5,6,7-trihydro-11-methoxybenzo, a melatonin receptor antagonist c) 1 uM of cyclohept [2,1-a] indol-13-yl) ethanamine was added to fresh medium daily over about 5-7 days. As a control, MEF medium, which is a medium for inducing differentiation, was used.

In order to confirm the differentiation of the stem cells, the nuclear staining with DAPI was confirmed the presence and distribution of cells. In addition, to distinguish the undifferentiated cells, the degree of staining of human stem cells was examined using an antibody to Oct4 (anti mouse, Santa Cruz # sc5279, U.S.A.), which is an undifferentiated label (red fluorescence). First, the cells were fixed with 4% para-formaldehyde solution, and then washed three times with 1x PBST (1xPBS, 0.05% Tween-20) three times for 5 minutes. After blocking for 30 minutes with 10% normal goat serum, the cells were reacted with the primary antibody OCT4 at room temperature for 1 hour. After washing 3 times with 1x PBST for 10 minutes, it is reacted with fluorescent secondary antibody for 45 minutes. After washing twice with 1x PBST, DAPI was treated for 5 minutes to stain nuclei and washed twice more with 10 times each with 1x PBST.

As a result, 1 uM N-butanoyl 2- (5,6,7-trihydro-11-methoxybenzo () in culture conditions (FIG. 2A) to maintain the undifferentiated state of human embryonic stem cells ( c) Treatment with cyclohept [2,1-a] indol-13-yl) ethanamine resulted in the loss of stem cell nature and differentiation of human embryonic stem cells as shown in (b) and (e) of FIG. 2. (Cells spread around).

As shown in (c) and (f) of FIG. 2, when the MEF medium, which is a differentiation-inducing medium used as a control, was used, colonies of human embryonic stem cells disappeared, and cells were spread widely. Will not be expressed.

1 is a cell staining photograph showing that the melatonin receptor agonist inhibits the differentiation of human embryonic stem cells and maintains stem cellity.

Figure 2 is a cell staining picture showing that melatonin receptor antagonists induce differentiation of human embryonic stem cells.

Claims (14)

A medium composition for maintenance of stem cells of human embryonic stem cells comprising N- [2- (5-methoxy-1H-indol-3-yl) ethyl] acetamide, which is a melatonin receptor agonist in a basic medium. delete According to claim 1, wherein the melatonin receptor agonist is stem cell maintenance medium composition, characterized in that it comprises 0.001 to 1000 uM in the medium composition. The method of claim 1, wherein the basic medium is Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, α-MEM (α-Minimal). Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), MEF and mTeSR-1, characterized in that the medium composition for stem cell maintenance. delete delete Base medium contains melatonin receptor antagonist N-butanoyl 2- (5,6,7-trihydro-11-methoxybenzo (c) cyclohept [2,1-a] indol-13-yl) ethanamine A medium composition for inducing stem cell differentiation of human embryonic stem cells. 8. The method of claim 7, wherein the melatonin receptor antagonist is a medium composition for inducing stem cell differentiation, characterized in that it comprises 0.001 to 1000 uM in the medium composition. delete The method of claim 7, wherein the basic medium is Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, α-MEM (α-Minimal). Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Isocove's Modified Dulbecco's Medium), MEF and mTeSR-1, characterized in that the medium composition for stem cell differentiation induction. delete delete A method of culturing human embryonic stem cells while maintaining the characteristics of human embryonic stem cells using the medium composition according to any one of claims 1, 3 to 4. Clause 7, 8 And A method of inducing differentiation of human embryonic stem cells using the medium composition according to claim 10.

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